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Molecular determinants of cGMP binding to chicken cone photoreceptor phosphodiesterase.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2004 Nov 12; Vol. 279 (46), pp. 48143-51. Date of Electronic Publication: 2004 Aug 25. - Publication Year :
- 2004
-
Abstract
- Structural studies on photoreceptor phosphodiesterases type 6 (PDE6s) have been hampered by an inability to express and purify substantial amounts of enzyme. Here we describe bacterial expression and characterization of the chicken cone PDE6 regulatory GAF-A and GAF-B domains. High affinity cGMP binding was found only for GAF-A as predicted from sequence alignments with the GAF domains of PDE2 and PDE5. A homology model of the GAF-A domain of chicken cone PDE6 based on the crystal structure of mouse PDE2A GAF-B was used to identify residues likely to make contact with cGMP. Alanine mutagenesis of 4 of these residues (F123A, D169A, T172A, and T176A) showed that each was absolutely required for cGMP binding. Three of these residues map to the H4 helical structure of the GAF-A domain indicating this region as a key structural component for cGMP binding. Mutagenesis of another residue, S97A, decreased cGMP binding affinity 5-fold. Finally mutagenesis of Glu-124 indicated that it is responsible for part but not all of the high specificity for cGMP binding to PDE6 GAF-A. Since little data is available on the properties of the chicken cone PDE6 holoenzyme, we also characterized the native PDEs of chicken retina. Two histone-activated PDE6 peaks were separated by ion exchange chromatography and identified by mass spectrometry as cone and rod photoreceptor PDE6s, respectively. Both of these PDEs had cGMP binding and kinetic properties similar to their corresponding bovine photoreceptor PDEs. Moreover the cGMP binding properties of chicken cone PDE6 holoenzyme were very similar to those of the bacterially expressed individual GAF-A or GAF-A/B domains.
- Subjects :
- 3',5'-Cyclic-AMP Phosphodiesterases chemistry
3',5'-Cyclic-AMP Phosphodiesterases genetics
3',5'-Cyclic-AMP Phosphodiesterases metabolism
3',5'-Cyclic-GMP Phosphodiesterases chemistry
3',5'-Cyclic-GMP Phosphodiesterases isolation & purification
Amino Acid Sequence
Animals
Cattle
Chickens
Cyclic Nucleotide Phosphodiesterases, Type 2
Eye Proteins chemistry
Eye Proteins genetics
Eye Proteins isolation & purification
Eye Proteins metabolism
Mice
Models, Molecular
Molecular Sequence Data
Mutation
Protein Binding
Protein Structure, Tertiary
Protein Subunits chemistry
Protein Subunits genetics
Protein Subunits metabolism
Sequence Alignment
3',5'-Cyclic-GMP Phosphodiesterases genetics
3',5'-Cyclic-GMP Phosphodiesterases metabolism
Cyclic GMP metabolism
Protein Conformation
Retinal Cone Photoreceptor Cells enzymology
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 279
- Issue :
- 46
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 15331594
- Full Text :
- https://doi.org/10.1074/jbc.M404338200